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The loss of outer capsid protein P2 results in nontransmissibility by the insect vector of rice dwarf phytoreovirus.

ABSTRACT: A transmission-defective (TD) isolate of rice dwarf phytoreovirus lacked the ability to infect cells when derived from the virus-free insect vector Nephotettix cincticeps. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified virus showed that among six structural proteins, the P2 outer capsid protein (encoded by genome segment S2) was absent from the TD isolate, whereas all six proteins were present in the transmission-competent (TC) isolate. P2 was not detected on immunoblots of rice plants infected with the TD isolate. Genome segment S2 and its transcript were detected in both TD and TC isolates. Sequence analysis of the S2 segment of the TD isolate revealed the presence of a termination codon due to a point mutation in the open reading frame, which might explain the absence of P2 in the TD isolate. These results demonstrate that the P2 protein is one of the factors essential for infection by the virus of vector cells and, thus, influences transmissibility by vector insects.

SUBMITTER: Tomaru M

PROVIDER: S-EPMC192165 | biostudies-other | 1997 Oct

REPOSITORIES: biostudies-other

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The loss of outer capsid protein P2 results in nontransmissibility by the insect vector of rice dwarf phytoreovirus.

Tomaru M M Maruyama W W Kikuchi A A Yan J J Zhu Y Y Suzuki N N Isogai M M Oguma Y Y Kimura I I Omura T T

Journal of virology 19971001 10

A transmission-defective (TD) isolate of rice dwarf phytoreovirus lacked the ability to infect cells when derived from the virus-free insect vector Nephotettix cincticeps. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified virus showed that among six structural proteins, the P2 outer capsid protein (encoded by genome segment S2) was absent from the TD isolate, whereas all six proteins were present in the transmission-competent (TC) isolate. P2 was not detected ...[more]

PMID: 9311898

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Project description:BackgroundSouthern rice black-streaked dwarf virus (SRBSDV) has spread from the south of China to the north of Vietnam in the past few years, and has severely influenced rice production. However, previous study of traditional SRBSDV transmission method by the natural virus vector, the white-backed planthopper (WBPH, Sogatella furcifera), in the laboratory, researchers are frequently confronted with lack of enough viral samples due to the limited life span of infected vectors and rice plants and low virus acquisition and inoculation efficiency by the vector. Meanwhile, traditional mechanical inoculation of virus only apply to dicotyledon because of the higher content of lignin in the leaves of the monocot. Therefore, establishing an efficient and persistent-transmitting model, with a shorter virus transmission time and a higher virus transmission efficiency, for screening novel anti-SRBSDV drugs is an urgent need.MethodsIn this study, we firstly reported a novel method for transmitting SRBSDV in rice using the bud-cutting method. The transmission efficiency of SRBSDV in rice was investigated via the polymerase chain reaction (PCR) method and the replication of SRBSDV in rice was also investigated via the proteomics analysis.ResultsRice infected with SRBSDV using the bud-cutting method exhibited similar symptoms to those infected by the WBPH, and the transmission efficiency (>80.00%), which was determined using the PCR method, and the virus transmission time (30 min) were superior to those achieved that transmitted by the WBPH. Proteomics analysis confirmed that SRBSDV P1, P2, P3, P4, P5-1, P5-2, P6, P8, P9-1, P9-2, and P10 proteins were present in infected rice seedlings infected via the bud-cutting method.ConclusionThe results showed that SRBSDV could be successfully transmitted via the bud-cutting method and plants infected SRBSDV exhibited the symptoms were similar to those transmitted by the WBPH. Therefore, the use of the bud-cutting method to generate a cheap, efficient, reliable supply of SRBSDV-infected rice seedlings should aid the development of disease control strategies. Meanwhile, this method also could provide a new idea for the other virus transmission in monocot.

Project description:The discovery that adeno-associated virus 2 (AAV2) encodes an eighth protein, called assembly-activating protein (AAP), transformed our understanding of wild-type AAV biology. Concurrently, it raised questions about the role of AAP during production of recombinant vectors based on natural or molecularly engineered AAV capsids. Here, we show that AAP is indeed essential for generation of functional recombinant AAV2 vectors in both mammalian and insect cell-based vector production systems. Surprisingly, we observed that AAV2 capsid proteins VP1 to -3 are unstable in the absence of AAP2, likely due to rapid proteasomal degradation. Inhibition of the proteasome led to an increase of intracellular VP1 to -3 but neither triggered assembly of functional capsids nor promoted nuclear localization of the capsid proteins. Together, this underscores the crucial and unique role of AAP in the AAV life cycle, where it rapidly chaperones capsid assembly, thus preventing degradation of free capsid proteins. An expanded analysis comprising nine alternative AAV serotypes (1, 3 to 9, and rh10) showed that vector production always depends on the presence of AAP, with the exceptions of AAV4 and AAV5, which exhibited AAP-independent, albeit low-level, particle assembly. Interestingly, AAPs from all 10 serotypes could cross-complement AAP-depleted helper plasmids during vector production, despite there being distinct intracellular AAP localization patterns. These were most pronounced for AAP4 and AAP5, congruent with their inability to rescue an AAV2/AAP2 knockout. We conclude that AAP is key for assembly of genuine capsids from at least 10 different AAV serotypes, which has implications for vectors derived from wild-type or synthetic AAV capsids.IMPORTANCE Assembly of adeno-associated virus 2 (AAV2) is regulated by the assembly-activating protein (AAP), whose open reading frame overlaps with that of the viral capsid proteins. As the majority of evidence was obtained using virus-like particles composed solely of the major capsid protein VP3, AAP's role in and relevance for assembly of genuine AAV capsids have remained largely unclear. Thus, we established a trans-complementation assay permitting assessment of AAP functionality during production of recombinant vectors based on complete AAV capsids and derived from any serotype. We find that AAP is indeed a critical factor not only for AAV2, but also for generation of vectors derived from nine other AAV serotypes. Moreover, we identify a new role of AAP in maintaining capsid protein stability in mammalian and insect cells. Thereby, our study expands our current understanding of AAV/AAP biology, and it concomitantly provides insights into the importance of AAP for AAV vector production.

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The loss of outer capsid protein P2 results in nontransmissibility by the insect vector of rice dwarf phytoreovirus.

Publications

The loss of outer capsid protein P2 results in nontransmissibility by the insect vector of rice dwarf phytoreovirus.

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